Certain aberrations in the neurochemical circuitry of the brain's basal ganglia are known to result in Parkinson's disease. When dopamine neurons die (the primary pathology in Parkinson's disease), the loss of dopamine to the striatum (caudate nucleus and putamen) sets into motion a series of changes in the neural activity of other brain nuclei located downstream from the striatum. FIG. 1 shows the normal neurochemical circuitry of the brain's basal ganglia.
Current neurosurgical approaches to this problem include either destroying the internal globus pallidus (GPi) by pallidotomy or implanting electrodes for electrical stimulation of this region of the brain. There are major disadvantages to these current neurosurgical approaches. Pallidotomy is permanent; can have serious side effects depending on the precision of the lesion; and can result in dementia and other problems due to destruction of fibers of passage going through the GPi en route to other structures. Electrical stimulation procedures require implantation of electrodes in the brain and a control box under the skin. Over time, a tissue reaction could occur, thereby rendering electrodes useless or dysfunctional. Moreover, chronic electrical stimulation can damage tissue, and epileptic foci kindling may develop as a result of chronic electrical stimulation.
Traditional pharmacotherapies also have serious drawbacks in that the neurochemicals that need to be down-regulated in target structures are ubiquitous in the brain. Systemic administration of inhibitors of these neurochemicals can cause seizures, psychosis, coma, and even death.
The current invention has a number of novel features which represent improvements over the current treatments for Parkinson's disease. In the present invention, molecular neurosurgery is utilized. This approach, through the use of specific antisense or triplex oligonucleotides obviates the problems presented by conventional neurosurgical and pharmacotherapeutic approaches. Via the molecular neurosurgery described more fully below, the current invention selectively alters the functional status of specific groups of neurons without interfering with the functioning of other nearby neurons. This selective approach is made possible by targeting the application of the oligonucleotides to specific neurochemicals in specific neural structures. More precisely, selective targeting occurs by introducing antisense or triplex expression vectors to provide more long-term changes in gene expression. In this way, the result is selective inhibition of the abnormal functioning of certain neural circuits in the Parkinsonian brain without interfering with the normal functioning of these neurochemicals in the rest of the brain. Additionally, by altering the concentration or sequence of the oligonucleotides, the invention makes it possible to titrate the degree to which abnormal activity is inhibited in the target circuits. This ability to titrate further ensures that the treatment only interferes with abnormal functions and does not cause undesirable side effects.
The current invention uses a molecular biological method of treating Parkinson's disease. The antisense oligonucleotides of the present invention are short sequences of phosphorothioate nucleotides, designed in the laboratory. These oligonucleotides block the translation of messenger RNA (mRNA) into protein at the ribosome. (FIG. 2).
Another way to prevent or decrease the expression of deleterious genes is to block transcription of DNA. This approach is also utilized in the present invention. Oligonucleotides, designed in the laboratory, form triplex structures and block the transcription site on target DNAs.
Antisense or triplex oligonucleotides are targeted to the internal globus pallidus and/or substantia nigra pars reticulata (SNr) where the expression of glutamic acid decarboxylase (GAD67, GAD65, or a combination of the two isoforms) is downregulated; Glutamic acid decarboxylase (in the form of GAD67, GAD65, or a combination of the two isoforms) is the synthesizing enzyme for the production of the inhibitory neurotransmitter gamma aminobutyric acid (GABA). As a result of glutamic acid decarboxylase downregulation, a decrease occurs in the abnormally high output of GABA by the GPi and the SNr, and the symptoms of Parkinson's disease are ameliorated.
Other targets for the antisense or triplex oligonucleotide treatment of the present invention are the glutamate receptors on neurons in the GPi and the SNr. Decreased expression of these receptors blunts the response of these neurons to increased levels of glutamate released by projections from the subthalamic nucleus (STN) to the GPi and the SNr. (FIG. 3). Another potential target for the antisense or triplex oligonucleotide treatment of the present invention are GABA receptors on neurons in the thalamic motor nuclei that receive excess inhibitory inputs from the GPi and the SNr.
Overall, the present invention is a treatment for Parkinson's disease, which is carried out by administering antisense oligonculeotides directed to the transcripts encoding the synthesizing enzyme GAD, thereby leading to a highly selective blockade of GABA. Although the actions of the antisense are selective, they will be relatively short-lived. According to the literature, systems return to normal within a few days after cessation of treatment with antisense oligonculeotides. Typically, in order to demonstrate an effect on protein production, antisense oligonucleotides need to be administered repeatedly or by continuous infusion. As a result, the present invention employs RNA expression vectors. These eucaryotic expression vectors producing RNA antisense to GAD65 or GAD67 are injected into the desired brain region to transfect neurons so that when the cells receive a signal to produce GABA, the antisense would be activated and GABA is not produced.